This proposal seeks to unravel unresolved questions in the mechanisms and regulation of prokaryotic transcription initiation, elongation and termination, as well as in early stages of eukaryotic transcription initiation. Single molecule fluorescence spectroscopic and microscopic methods will be used to detail the sequence, magnitude, kinetics and order of conformational transitions and intermediates along the basal transcription cycle pathway. Transient protein-DNA and protein-protein interactions (and their induced conformations) of the prokaryotic transcription machinery's subunits and of some of the eukaryotic transcription machinery's subunits will be investigated. The mechanisms of prokaryotic and eukaryotic transcription regulation will also be explored using several transcription factors as model systems. Several single molecule assay formats, implemented on freely diffusing transcription complexes and on immobilized complexes will be utilized to address mechanistic and kinetic questions respectively. The proposed studies are expected to elucidate the role of conformational transitions and biomolecular interactions in transcription and its regulation, and the tools developed for these studies could be generalized for the study of regulatory circuits and other biomolecular machineries. The proposed studies of prokaryotic transcription will provide a basis for understanding antibacterial drugs (through inhibition of conformations) and therefore will provide relevant insight and assays for drugs design and screening. The proposed studies of eukaryotic transcription will lay a foundation for understanding mechanisms of transcription related human diseases and will aid in designing and screening therapeutics agents for these diseases. PUBLIC HEALTH RELEVANCE: The proposed work will unravel the detailed mechanism of the first and most important step in gene expression, i.e. transcription and transcription regulation. It will pave the way for detailed molecular and structural understanding of the action of antibacterial agents. It will also provide answers for several long standing and unresolved questions regarding the early stages of eukaryotic transcription initiation and regulation. The latter will provide detailed molecular understanding of the causes for transcription related diseases and will provide tools for drug design and screening that will prevent, manage and cure these diseases.